In the twentieth century the treatment of infectious diseases was revolutionized by the development of antibiotics. However, due to their widespread use resistance to antibiotics is increasing on a global scale, such that adequate therapies are lacking for both previously controlled and emerging bacterial diseases. Moreover, the molecular targets and mechanisms of action of most newly developed antibiotics are similar to current ones, reducing their efficacy in the face of resistance. The effective treatment of infectious diseases in the face of increasing antibiotic resistance requires the development of pharmaceuticals that act upon novel conserved targets.
Bacterial virulence properties are viable targets for the development of novel therapeutic agents because such agents would not kill bacteria themselves but block disease. Because these agents would not inhibit general bacterial growth as part of their mechanism of action, this strategy could decrease the likelihood for selection of resistance and reduce side effects by sparing commensal organisms. However, many pathogenic mechanisms are microbial specific, necessitating more rapid pathogen identification than currently is in clinical practice to be useful. Furthermore, a restricted spectrum of activity would decrease the economic incentive necessary for their development.
In the case of many self-limiting enteric infections, the use of traditional antibiotics may prolong colonization and increase toxin release and, therefore, antibiotic treatment would not be recommended. Furthermore, compounds that target virulence properties may be particularly relevant for biodefense in which organisms resistant to traditional antibiotics may be generated by genetic engineering. Engineering organisms resistant to anti-virulence agents may not be as straightforward, as organisms resistant to antibiotics can be generated by selection for bacterial growth, but such a selection would not exist for reconstitution of bacterial virulence properties. In addition, for purposes of biodefense, there is a need for therapeutic agents with a broad spectrum of activity, because single treatments for single infectious agents present problems in terms of production, diagnosis, storage, and distribution.
Gram-negative bacterial virulence secretion systems are essential for a wide array of animal and plant infectious diseases. Two prominent examples of Gram-negative bacterial virulence associated secretion systems, termed type II secretion (T2S) and type III secretion (T3S), are responsible for the pathogenesis of many infectious diseases including plague, gastroenteritis, Gram-negative pneumonia, dysentery, enteric fever, tularemia, trachoma, endometritis, and a variety of plant diseases. T2S is also known as the terminal component of the General Secretory Pathway (GSP), because it is a two step process where substrates are secreted across the bacterial inner membrane by the GSP, also known as sec-dependent secretion and subsequently transported across the outer membrane. T2S systems secrete a variety of mammalian toxins as well as proteins, which degrade host cell components, such as proteins, lipids and sugars of the extracellular matrix. Interestingly, a number of the genes required for T2S are homologous to those required for type IV pilus (T4P) assembly on the cell surface of some bacteria. T4P are required for twitching motility, a flagella independent form of bacterial translocation, which plays a role in host colonization and biofilm formation in organisms, such as enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, Vibrio cholera and Neisseria gonorrhea. T3S systems are complex multi protein organelles that assemble in the bacterial membrane of more than 25 Gram-negative animal and plant pathogens to deliver multiple virulence proteins directly from the bacterial cytosol into host cells. These secreted proteins influence host cell physiology by altering a variety of antibacterial functions with resultant disease.
Unfortunately, many of the components of Gram-negative bacterial secretion systems are not well conserved among the various systems and would not make ideal drug targets, but one component the secretin, has a broadly conserved structure despite diverse amino acid sequence. The secretin protein associates into large and highly stable oligomeric complexes of 12-14 subunits in the outer membrane, which functions as an export channel for substrate secretion. In T3S systems these proteins form the outer membrane component of the needle complex (NC); a multi protein complex that is the transmembrane component of the complex T3S apparatus. Secretins are membrane spanning proteins that are synthesized in the bacterial cytoplasm and exported to the periplasm by the sec-dependent pathway. In many systems the secretin has a dedicated lipoprotein that appears to function to promote insertion and polymerization of the ring in the outer membrane. Secretin proteins have two major domains, which are approximately equal in length. The C terminal domain is well conserved and believed to anchor the protein in the membrane by 10 14 potentially transmembrane amphipathic β strains characteristic of other outer membrane proteins. In contrast, the N terminal domain is much less conserved and believed to facilitate recognition of substrates and confer secretion specificity. Finally, there is occasionally a third domain present downstream from the C terminal domain, which interacts with the lipoprotein to facilitate localization to the outer membrane.
There exists a need for inhibitors of type III secretion in Gram-negative bacteria. The present invention fulfills this need, and provides further related advantages.